Veterinary Parasitology 202 (2014) 248–256

Contents lists available at ScienceDirect

Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar

Seroprevalence of Babesia caballi and Theileria equi in five draught equine populated metropolises of Punjab, Pakistan Muhammad Hammad Hussain a,∗ , Muhammad Saqib a , Fahad Raza b , Ghulam Muhammad a , Muhammad Nadeem Asi a , Muhammad Khalid Mansoor a , Muhammad Saleem c , Abdul Jabbar d a b c d

Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan Faculty of Veterinary and Animal Science, PMAS Arid Agriculture University, Rawalpindi, Pakistan The Brooke Hospital for Animals, Faisalabad, Pakistan Faculty of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia

a r t i c l e

i n f o

Article history: Received 16 June 2013 Received in revised form 25 January 2014 Accepted 28 January 2014

Keywords: Seroprevalence Piroplasmosis Theileria equi Babesia caballi Draught equines cELISA Pakistan

a b s t r a c t Equine piroplasmosis (EP) caused by intraerythrocytic parasites (Theileria equi and Babesia caballi) is an emerging equine disease of world-wide distribution. In Pakistan, the prevalence and incidence of EP are unknown. In order to obtain the first insights into the prevalence of the disease, a total of 430 equids, including 33 mules, 65 horses and 332 donkeys, aging from ≤5 to ≥10 years of either sex, from five metropolises of Punjab, Pakistan, were serologically tested for the presence of antibodies directed against B. caballi and T. equi, using a competitive enzyme-linked immunosorbent assay (cELISA). Out of 430 equid serum samples tested, 226 (52.6%, 95% CI 47.7–57.4) were found cELISA positive for EP (T. equi and/or B. caballi infections). The overall seroprevalence of EP was 41.2% (95% CI 36.5–46.0) for T. equi and 21.6% (95% CI 17.8–25.8) for B. caballi. A small proportion of equids (10.2%, 95% CI 7.5–13.5) was seropositive for both T. equi and B. caballi. Seroprevalence of T. equi was significantly higher (P < 0.01) in equines from the metropolis of Lahore (66.7%, 95% CI 54.3–77.6) and in horses (56.9%, 95% CI 44.0–69.2). Multivariable logistic regression model analysis indicated that factors associated with prevalence of EP were being an equine species kept in metropolis Lahore (OR = 4.24, 95% CI 2.28–7.90), horse (OR = 2.82, 95% CI 1.53–5.20) and male equids (OR = 1.81, 95% CI 1.15–2.86). © 2014 Elsevier B.V. All rights reserved.

1. Introduction Piroplasms (Apicomplexa: Piroplasmida) are ticktransmitted protozoa found in many wild and domestic animals. Equine piroplasmosis (EP), caused by intraerythrocytic parasites Babesia caballi (Nuttall and Strickland, 1910) and/or Theileria equi (Mehlhorn and Schein, 1998), is an important disease of solipeds (horses, donkeys, mules and zebras) worldwide (Kuttler, 1988). EP is mainly

∗ Corresponding author. Tel.: +92 41 9200161. E-mail address: [email protected] (M.H. Hussain). 0304-4017/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2014.01.026

transmitted by ticks belonging to three genera, including Dermacentor, Rhipicephalus and Hyaloma (Thompson, 1969; Jongejan and Uilenberg, 2004); however, sometimes iatrogenic transmission of the disease can also occur through contaminated blood transfusion, injections and surgical instruments (de Waal and van Heerden, 2004; Uilenberg, 2006; Vial and Gorenflot, 2006). The disease is endemic in Asia and many parts of Europe, south and central Americas and Africa (OIE, 2008). As per the data from World Animal Health Information Database (WAHID) Interface, 2013, 30 countries reported the occurrence of EP in the year 2012. Clinically, EP is usually characterized by fever, anorexia, depression, icterus, hemoglobinuria, bilirubinuria and

M.H. Hussain et al. / Veterinary Parasitology 202 (2014) 248–256

regenerative hemolytic anemia and, in some cases, death can also occur (Seifi et al., 2000; Camacho et al., 2005). However, clinical signs are not always overt, particularly in endemic areas because it is often difficult to recognise the infection as many factors (such as infecting dose, genetics of the infecting parasites and immune status of the host) contribute to the severity of the disease. T. equi infected animals usually exhibit a severe and acute form of EP; whereas, B. caballi infection usually follows a chronic course (Shkap et al., 1998; Vial and Gorenflot, 2006; Radostits et al., 2007). In addition, horses and donkeys may act as carriers of the parasite for several years (as in T. equi infection) or for the rest of their lives, so acting as reservoirs for ticks (Knowles, 1996; Rüegg et al., 2007; Kumar et al., 2009). Equines born and raised in the endemic areas enter into the carrier state of EP which can compromise their draught potential significantly (Abdelkebir et al., 2001) and these carriers are responsible for the maintenance of infection in endemic areas (Camacho et al., 2005). Heavy draught stress, strenuous exercise and poor nutrition can result into the recrudescent infection and clinical disease in these animals (Hailat et al., 1997; Seifi et al., 2000; Camacho et al., 2005). Severity of the clinical signs can vary depending on the host species involved. For instance, horses are more susceptible to B. caballi infections as compared to mules and donkeys (Acici et al., 2008) and susceptibility to the disease is directly proportional to the age of the animal (Abdelkebir et al., 2001). Traditionally, piroplasms are detected and identified by microscopic examination of thin blood smears prepared from acutely infected animals. A number of serological assays, including the complement fixation test (CFT), the indirect fluorescent antibody test (IFAT) and the enzyme linked immunosorbent assays (ELISAs) have been developed mostly for large scale epidemiological studies and to monitor infections during the latent stage of EP (Brüning et al., 1997; Shkap et al., 1998; Ikadai et al., 2000; de Waal and van Heerden, 2004). Later, direct detection methods using molecular tools (such as polymerase chain reaction) have been developed and are considered reliable diagnostic tools (Cacciò et al., 2000; Nagore et al., 2004). The competitive ELISA (cELISAs) employing monoclonal antibodies to recombinant antigens of T. equi (merozoites antigen 1; EMA-1) and B. caballi (rophtry associated protein; RAP-1) have been reported to be successful in detection of antibodies in sera of horses from different parts of the world (Knowles et al., 1992; Kappmeyer et al., 1999; Sevinc et al., 2008) and are currently tests of choice recommended by the World Organization for Animal Health (OIE, 2008). In Pakistan, very limited information is available on the status of EP in different species of draught equines (Kokab, 1986; Khan et al., 1987; Rashid et al., 2009) and these studies were based on conventional blood smear examination of T. equi and B. caballi, which does not always reveal the true picture of infections. The total equine population of Pakistan is ∼5.2 million (donkeys 4.6 million; horses 0.4; mules 0.2). From 1996 to 2006, the equine population increased in the country e.g., mules by 18%, donkeys by 20% and horses by 3% (Anonymous, 2006). Due to high fuel prices, marginalized communities use these animals as a cheap and economical means of transport that plays a

249

key role in the provision of livelihood for their owners. Any disease (such as EP) or disability rendering these animals sick and unavailable for work can have serious effects on owners and their families. Despite the importance of draught equines in Pakistan, we know very little about EP and its impact on these animals. Therefore, a cELISA-based cross-sectional serological survey was conducted to evaluate the level of exposure of draught equids (donkeys, horses and mules) to EP in five metropolises of Punjab Province of Pakistan. 2. Materials and methods 2.1. Study areas and meteorological characteristics A cross-sectional sero-epidemiological survey was conducted from July 2007 to March 2008 in five draught equine populated metropolises (Bahawalpur, Faisalabad, Gujranwala, Lahore and Multan) of Punjab, Pakistan. Geographic location and climatic characteristics of each metropolis included in the study are given in Table 1. Five districts included in the present study were selected based on the district equine population published in the Livestock Census of Pakistan (Anonymous, 2006). Wherever possible, the selection of study area(s) was made by taking into account the presence of operational areas of ‘Brooke Hospital for Animals’ (www.thebrooke.org.pk) in the selected cities to seek the necessary cooperation by the draught equine owners and convenient sampling. 2.2. Demography and husbandry characteristics of the study population According to the Pakistan Livestock Census (Anonymous, 2006), the total equine (donkeys, horses and mules) population in the study areas was 145,193 heads (Table 1). The highest number of equines dwelled in Faisalabad (53,835 heads), followed by Gujranwala (25,417), Lahore (22,703), Multan (21,970) and Bahawalpur (21,268) (Table 1) (Anonymous, 2006). Nondescript donkeys, horses and mules are used for pulling carts mainly for hauling industrial merchandize even in industrialized cities, particularly in Faisalabad which is famous for its textile industry. These animals are stall fed with zero grazing and alfalfa is the predominant fodder fed with supplementation of concentrate of chickpea (3–5 kg/day). Depending on the species involved, equines are usually put to work for 8–10 h/day for six days per week and haul 200–1500 kg per load. No immunization and mineral–vitamins supplementation program is in place. The animals are generally fed common salt along with Aloe vera and Leucas aspera, ranging from 100 to 300 g as a part of stimulant-carminative drench on weekly basis. 2.3. Sampling frame As the prevalence of EP was unknown in Pakistan, the sample size was calculated by considering the expected prevalence to be 50% with confidence limits of 95% and a desired absolute precision of 5% to collect maximum number of samples. The number of samples thus calculated was

250

M.H. Hussain et al. / Veterinary Parasitology 202 (2014) 248–256

Table 1 Coordinates, climatic characteristics and equine population of the five draught equine populated urban areas of Punjab, Pakistan included in the study. Metropolis

Bahawalpur Faisalabad Gujranwala Lahore Multan *

Coordinates

Elevation (from sea level – ft)

Latitude (◦ N)

Longitude (◦ E)

29◦ 31◦ 32◦ 31◦ 30◦

71◦ 71◦ 74◦ 74◦ 71◦

24 25 8 34 10

41 51 12 5 25

Summer ◦ C

370 600 744 712 710

Equine population*

Average temperature

Winter ◦ C

Maximum

Minimum

Maximum

Minimum

40 39 38 36 40.3

28 27 24 25 23

30 21 21 19 27

14 6 6 7 5.8

Donkey

Horse

Mule

19,076 44,144 18,964 17,386 19,076

1238 6645 4612 4596 1940

954 3046 1841 721 954

Total

21,268 53,835 25,417 22,703 21,970

Source: Anonymous (2006).

adjusted for finite population (Thrusfield, 2005) and 430 equine sera were collected. Stratification of the sampling units was performed by proportional allocation of sampling units to the draught equine population in the selected areas (Table 1). In each city, selection of sampling areas as well as animals on each location was made randomly. 2.4. Sample collection and animal data recording The present study was conducted after the approval of Research Synopsis Scrutiny Committee, Faculty of Veterinary Science, University of Agriculture, Faisalabad. The study period spanned over 8 months from July 2007 to March 2008. Blood samples (∼5 mL) were drawn into anticoagulant-free vacutainers and labeled accordingly. Following centrifugation, sera were harvested and preserved (−40 ◦ C) until tested. Data on the characteristics of the sampled animals (species, gender, age, location) management, history and treatment of EP were obtained on a pre-designed questionnaire completed by the investigators (M.H. Hussain and M. Saleem) on location during sampling. Physical and clinical examination of each animal sampled was performed and values regarding vital physiological parameters and observations were recorded. 2.5. Serological examination (cELISA) Commercially available cELISA kits (VMRD, Inc., Pullman, USA) for T. equi and B. caballi were used according to manufacturer’s instructions. These assays detect serum antibodies against recombinant T. equi merozoites antigen 1 (EMA-1), a surface protein on merozoites of T. equi (Knowles et al., 1992), and recombinant rhoptry-associated protein (RAP-1) of B. caballi (Kappmeyer et al., 1999). The optical density values were measured through an ELISA plate reader and the test results were obtained using the following formula:

 Percent Inhibition(%I) = 100 −

Sample O.D. × 100 Mean Negative Control O.D.



All samples producing ≥ 40% inhibition were regarded as positive whereas those with values < 40% were considered negative.

2.6. Statistical analyses Epidemiological data generated were analyzed by using software WINPEPI (Abramson, 2011). Chi-square testing was performed to find out significant difference among sex, age and locations based prevalence of T. equi and/or B. caballi. Odds ratio (OR) along with 95% CI were calculated for different potential determinants of the disease. The associations between the outcome response variables (seroprevalence of EP) and explanatory variables (information recorded on questionnaire) were estimated using binary logistic regression (IBM SPSS Statistics 17.0 for Windows® , IBM Corporation, Route 100 Somers, New York, USA). Individual animal was kept as a unit of analysis for determining significance of association. Outcome variables were dichotomized (0 = negative and 1 = positive) or categorized wherever applicable. Bivariable screening was conducted and variables yielding significant association (5.1–10 years >10 years

138 195 97

51 (36.9) 83 (42.6) 43 (44.3)

28.9–45.6 35.5–49.8 34.2–54.8

22 (15.9) 44 (22.6) 27 (27.8)

10.3–23.1 16.9–29.1 19.2–37.9

9 (6.5) 21 (10.8) 14 (14.4)

3.0–12.0 6.8–16.0 8.1–23.0

*

Values differ significantly (P < 0.05).

higher (P < 0.01) than those of other four metropolises. Prevalence of antibodies against B. caballi and mixed infection (B. caballi + T. equi) was not found significantly different in all 5 metropolises as well as among species of equines. No significant differences in infection levels between genders as well as age groups were observed for T. equi and/or B. caballi (Tables 3 and 4). However, male equines had higher odds of being seropositive (OR = 1.29) than females. Moreover, equines above 10 years of age (OR = 1.58) and those between 6 and 10 years of age (OR 1.38) were found more likely to test positive than equines aging less than or equal to five years (Table 4). 3.2. Association of seroprevalence of EP with epidemiological factors of the disease Epidemiological data collected were analyzed for any association between seroprevalence of infection and different variables (Table 4). Out of 226 seropositive equines, 105 (46.5%) were kept alone, 59 (26.1%) were managed with other equine cohorts and 62 (27.4%) equines were kept with animals other than equines (cattle, buffalo, sheep, goat and dog). Univariate analysis indicated that equines managed alone (OR = 0.83) or with equine cohorts (OR = 0.68) were less likely to be positive for EP as compared to those managed with non-equine cohorts. Presence of ticks was observed in cohorts of 71 equines (16.5%). Out of these, 38 (53.5%) were found positive for EP and 33 (46.5%) were seronegative. Odds of being seropositive for EP were higher in equines living with tick infested equine cohorts as compared to those kept with tick infested non-equine cohorts (OR = 2.04) and kept alone or with cohorts having no ticks (OR = 1.71). However, chances of being seropositive were found almost identical for equines living in tick infested housing (OR = 1.05) and apparently tick free housing (Table 4). Only owners of 111 (25.8%) equines reported the use of tick control measures and of these, 51 (45.9%) equines were seropositive for EP. Whereas, this figure was 54.8% (n = 175) for those equines whose owners practised no tick control methods. Equines belonging to the owners who did not use

tick control measures appeared to be more likely to test positive for EP (OR = 1.43) (Table 4). A significant difference was observed regarding the seroprevalence among equines of Lahore when compared for the tick control measures used or not. A significant difference was also observed regarding the seroprevalence of EP between Lahore and other areas when compared for the tick control used. One hundred veterinarians and auxiliary animal health workers (20 from each metropolis) were also interviewed for their knowledge and understanding about recommended treatments for B. caballi and T. equi. Only 12 veterinarians and auxiliary animal health workers had awareness about difference in recommended treatment for both parasites and tried to use recommended imidocarb dose for T. equi treatment (4 mg/kg, 4 times at 72 h intervals) in suspected cases. While, 88 preferred to use safer dose (2.2 mg/kg, two treatments at 24 h intervals) which was only adequate for B. caballi infections. Only 30% of these respondents reported to have examined the stained blood smears before starting the treatment in suspected cases. 3.3. Multivariable analysis for the prevalence of EP For the initial bivariable analysis, six variables were used, including metropolis, equine species, sex, age, presence of ticks in housing or cohorts, and tick control measures used. Analysis indicated that variables, including metropolis (P < 0.001), equine species (P 0.001), sex (P = 0.010) and age (P = 0.176) were significant enough (Wald P ≤ 0.2) to be included in multivariable analysis. However, presence of ticks in housing or cohorts, use of tick control measures and age were deleted from the model at subsequent steps (P > 0.05) and the resulting final model suggested that prevalence of EP in various equine species might vary based on the different metropolis and sex. Hosmer–Lemeshow test (2 = 11.96, 7df, P = 0.102) and Nagelkerke R2 square (0.134) values suggested that this model was good to fit. For further analysis, categorical variables (metropolis and equine species) were collapsed and tested for interaction term. For this purpose, two new variables were created one comparing the prevalence

M.H. Hussain et al. / Veterinary Parasitology 202 (2014) 248–256

253

Table 4 Risk factors for predicting EP (caused by Theileria equi and/or Babesia caballi) in equines (n = 430) of five draught equine populated metropolises of Punjab, Pakistan. Factors Metropolis

Equine species

Sex Age

Ticks on cohorts

Housing Tick control

Multan Bahawalpur Faisalabad Gujranwala Lahore Horse Mule Donkey Male Female ≤5 years >5–10 years >10 years Alone/tick negative cohorts Tick positive non equine cohorts Ticks on equine cohorts Tick infested Without ticks Not used Used

Seroprevalence (%)

Odds ratio (confidence interval 95%)

P value

45.6 44.1 52.7 42.7 78.3 69.2 63.6 48.2 54.6 48.2 46.4 54.4 57.7 52.4 47.9 65.2 53.5 52.4 54.9 45.9

0.23 (0.11–0.49) 0.22 (0.10–0.46) 0.31 (0.16–0.60) 0.21 (0.10–0.43) 1.0 2.42 (1.37–4.27) 1.88 (0.90–3.95) 1.0 1.29 (0.86–1.95) 1.0 0.63 (0.38–1.07) 0.87 (0.53–1.43) 1.0 0.59 (0.24–1.42) 0.49 (0.18–1.37) 1.0 1.05 (0.63–1.74) 1.0 1.43 (0.93–2.21) 1.0

0.00

of EP in Lahore with all other metropolises and other comparing the prevalence in horses with mules and donkeys. The model was re-run with these new variables along with sex and interaction term to assess the inference that effect of metropolis varies by equine species. Inclusion of the interaction term improved the model fitness (Hosmer–Lemeshow test 2 = 0.461 and Nagelkerke R2 = 0.112) and indicated that males (OR = 1.81, 95% CI 1.15–2.86), equines of metropolis Lahore (OR = 4.24, 95% CI 2.28–7.90) and horses (OR = 2.82, 95% CI 1.53–5.20) were more likely to be test positive for EP (Table 5).

0.003

0.215 0.182

0.387

0.859 0.105

seroprevalence of EP (caused by T. equi and/or B. caballi) using cELISA in draught equines from Pakistan. In the present study, the overall seroprevalence of EP was 52.6% for all piroplasms, 41.2% for T. equi, 21.6% for B. caballi and 10.2% for mixed infections of these parasites. The seroprevalence of EP was significantly higher (P < 0.01) in equines from the metropolis of Lahore than those from other metropolises. Previously, a number of studies have reported a wide range (0–100%) of seroprevalence for EP using various serological tests (Barbosa et al., 1995; Ribeiro et al., 1999; Akkan et al., 2003; Boldbaatar et al., 2005; Karatepe et al., 2009). The difference in the prevalence of EP among countries and even in different climatic zones within a country may be due to variations in sensitivity of the diagnostic tests employed, difference in the number and prevalence of vectors, activity of equids and the presence and effectiveness of control programs (Shkap et al., 1998; Asgarali et al., 2007; Karatepe et al., 2009; GarciaBocanegra et al., 2013). In the present study, the prevalence of EP in equines form Lahore could be attributed to significantly lower (P < 0.01) use of the tick control measures than other metropolises. Another possible reason for this higher seroprevalence in Lahore could be the presence of more carrier animals in the area as equines born and raised in endemic areas usually enter into the carrier state of EP (Abdelkebir et al., 2001; Camacho et al., 2005). We found that the seroprevalence of T. equi was higher than B. caballi. Previously, a number of studies have also reported that the prevalence of T. equi infection is usually

4. Discussion Piroplasmosis caused by T. equi and/or B. caballi is an important disease of equines and can lead to serious health and economic impacts. Over the years, the diagnosis of EP remained a challenge for the researchers and veterinarians as the peripheral blood stained (Giemsa or Leishman) smears are rarely conclusive and most of the serological tests have problems, including reporting of false positive/negative results (Donnelly et al., 1980; Ribeiro et al., 1999; Abdelkebir et al., 2001; Akkan et al., 2003; Zinora et al., 2007). cELISA used in the present study has been recognized by OIE and various studies have reported its usefulness (Knowles et al., 1991; Shkap et al., 1998; Katz et al., 2000; Balkaya et al., 2010; Jaffer et al., 2010; GarciaBocanegra et al., 2013). This is the first report on the

Table 5 Multivariable logistic regression analysis for predicting piroplasmosis in draught equines (n = 430) from five metropolises of Punjab, Pakistan. Exposure variable

Comparison

Odds ratio

Metropolis Lahore Horses Male

All other metropolises Mules and donkeys Female

4.24 2.82 1.81

95% CI

P value

Lower

Upper

2.28 1.53 1.15

7.90 5.20 2.86

0.000 0.001 0.010

254

M.H. Hussain et al. / Veterinary Parasitology 202 (2014) 248–256

higher in the endemic regions of the world, including Brazil (Barbosa et al., 1995; Ribeiro et al., 1999), Japan (Ikadai et al., 2002), Mongolia (Boldbaatar et al., 2005), Spain (Camacho et al., 2005; Garcia-Bocanegra et al., 2013), Trinidad (Asgarali et al., 2007) and Turkey (Akkan et al., 2003; Karatepe et al., 2009). The higher seroprevalence of T. equi as noted in this study could be ascribed to the fact that T. equi infections are usually life-long, whereas infections with B. caballi usually subside in 4–5 years (Shkap et al., 1998; Vial and Gorenflot, 2006; Rüegg et al., 2007; Rüegg et al., 2008). In addition, B. caballi infection clears away from the body in few years and generally responds well to the recommended dosage of imidocarb treatment (Brüning, 1996; Ali et al., 1996; Rüegg et al., 2007; Rüegg et al., 2008). However, some workers have also reported insignificant differences in the prevalence of T. equi and B. caballi (Shkap et al., 1998; Heuchert et al., 1999; Xuan et al., 2001; Chahan et al., 2006; Torina et al., 2007). One of the limitations of this study was that only small number of horse (21%) and mule (32%) aging ≤5 years were sampled and this could have contributed to the higher prevalence of T. equi. Moreover, the selection of target equine population may also have resulted in higher seroprevalence of T. equi herein because we selected those equids that were kept for draught purpose only and were usually kept under poor management, improper housing and higher draught stress, hence increasing the chance of acquiring EP. Seroprevalence of T. equi infection in horses was significantly higher (P < 0.01) as compared to donkeys and mules. Possible reason for this could be that the horses have less vigor and strength to bear the heavy draught burden than mules and donkeys. Chahan et al. (2006) from China and Torina et al. (2007) from Italy reported that the seroprevalence of B. caballi infection was higher in donkeys than in horses. However, higher seroprevalence of EP was observed in mules as compared to horses in Greece (Kouam et al., 2010) and Spain (Garcia-Bocanegra et al., 2013). There was no association found between seroprevalence of antibodies against B. caballi and equine species (P = 0.33) and a similar finding was reported by Acici et al. (2008) from Turkey. Only numerical differences were found possibly due to the differences in the sampled proportions of these equines. Seroprevalence of T. equi and B. caballi antibodies observed in this study was not found significantly (P > 0.05) associated with age of equines. However, prevalence of antibodies against both parasites increased with the age of equines and more prevalence was observed in older equines (>5 years of age) as compared to young ones (≤5 years of age). Various authors (Brüning, 1996; Asgarali et al., 2007; Rüegg et al., 2007; Rüegg et al., 2008; Karatepe et al., 2009; Kouam et al., 2010) have reported in their respective studies that once infected with T. equi, equines remain carriers throughout their life and this could be a possible reason for the higher seropositivity in older equines. The seroprevalence of EP was not found associated with sex (P > 0.05) and this is in accord with that reported by Asgarali et al. (2007), Karatepe et al. (2009) and Sigg et al. (2010). However, Shkap et al. (1998) and Rüegg et al. (2007) found a higher seropositivity in mares and geldings as compared to that in stallions which could be due to the different level of care, grooming and attention these animals were

getting according to their importance for owners. In the present study, the numerically higher seropositivity found in males as compared to females could be due to the large proportion of male equines enrolled for sampling as compared to females. In the present study, no significant association (P > 0.05) was found between housing pattern and seroprevalence of EP. Computed odds ratio indicated that equines living alone (OR = 0.83)/with equine cohorts (OR = 0.68) in these metropolises were less likely to be positive for EP as compared to those managed with other domestic animals and kept for the domestic draught purposes (ploughing or carrying fodder for other animals). Similarly, a few authors found that the risk of contracting EP were higher in equines if they had greater interaction with each other or other domestic animals (Heuchert et al., 1999; Camacho et al., 2005 and Karatepe et al., 2009; Garcia-Bocanegra et al., 2013) and also in those being used for farm work that increases the likelihood of greater tick infestation, possibly due to an access to pastures (Kouam et al., 2010 and Abutarbush et al., 2012). Although we found a positive association between the presence of ticks on equines or non-equine cohorts and the seroprevalence of EP, chances of being seropositive were found almost identical for equines living in tick-infested or -free housings. Similarly, the seroprevalence of EP was found to be higher in those equines and areas where owners were not giving much emphasis on vector control measures (OR = 1.43). These findings are concordant with those reported by Heuchert et al. (1999), Barbosa et al. (1995), Abdelkebir et al. (2001) and Chahan et al. (2006), who noticed a positive correlation between EP and tick infestation in equines and their equine or non-equine cohorts. However, ticks were not collected from equines during this study and there is still a need to establish the competent tick vector responsible for the spread of EP in Pakistan. The structured interview of practicing veterinarians and animal health workers indicated that lacunae regarding the correct diagnosis and treatment of EP existed and a majority relied upon clinical signs for making the presumptive diagnosis of the disease and the practice of using the results of microscopic examination of Giemsa stained smears and other serological tests was almost non-existent. Majority of veterinarians and animal health workers used treatment protocol recommended for B. caballi (imidocarb dipropionate at 2.2 mg/kg body weight, twice at 24 hour interval) to treat all suspected cases and that might have led to a higher seroprevalence of T. equi found in this study. Conducting workshops, lectures and seminars regarding EP, its diagnosis and correct treatment as reported by several authors (Ali et al., 1996; Hailat et al., 1997; Seifi et al., 2000; Vial and Gorenflot, 2006) can address this issue in future. 5. Conclusions This study first time presents the seroprevalence of EP in draught equines from Pakistan and indicates that EP is endemic in equines of the studied metropolises of Pakistan. In order to understand the epidemiology of disease and devise control measures, future studies by employing conventional and molecular techniques are required to identify

M.H. Hussain et al. / Veterinary Parasitology 202 (2014) 248–256

the competent tick vectors along with seasonal distribution and other factors responsible for the spread and maintenance of EP in the region. Veterinarians and animal health workers should administer the standard treatment regimen(s) recommended for each type of piroplasm (T. equi or B. caballi) after proper diagnosis to control the disease. Where a lack of proper diagnostic facilities precludes the diagnosis of the specific type of EP, the use of higher dose (i.e., 4 mg/kg as recommended for T. equi) is warranted. Conflict of interest None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgement The authors are extremely thankful to the Brooke Hospital for Animals, Pakistan for facilitating the field sampling in the communities of Faisalabad, Gujranwala, Lahore and Multan metropolises. References Abdelkebir, R., Sahibi, H., Lasri, S., Johnson, W.C., Kappmeyer, L.S., Hamidouch, A., Knowles, D.P., Goff, W.L., 2001. Validation of a competitive enzyme-linked immunosorbent assay for diagnosing Babesia equi infections of Moroccan origin and its use in determining the seroprevalence of B. equi in Morocco. J. Vet. Diagn. Invest. 13, 249–251. Abramson, J.H., 2011. WINPEPI updated: computer programs for epidemiologists, and their teaching potential. Epidemiol. Perspect. Innov. 8, 1, http://dx.doi.org/10.1186/1742-5573-8-1 Abutarbush, S.M., Alqawasmeh, D.M., Mukbel, R.M., Al-Majali, A.M., 2012. Equine babesiosis: seroprevalence, risk factors and comparison of different diagnostic methods in Jordan. Transbound. Emerg. Dis. 59, 72–78. Acici, M., Umur, S., Guvenc, T., Arslan, H.H., Kurt, M., 2008. Seroprevalence of equine babesiosis in the Black Sea region of Turkey. Parasitol. Int. 57, 198–200. Akkan, H.A., Karaca, M., Tutuncu, M., Deger, S., Keles, I., Agaoglu, Z., 2003. Serologic and microscopic studies on babesiosis in horses in the eastern border of Turkey. J. Eq. Vet. Sci. 23, 181–183. Ali, S., Sugimoto, C., Onuma, M., 1996. Equine piroplasmosis. J. Eq. Vet. Sci. 7, 67–77. Anonymous, 2006. Pakistan Livestock Census. Agriculture Census Organization, Government of Pakistan, Statistics Division. Asgarali, Z., Coombs, D.K., Mohammed, F., Campbell, M.D., Caesar, E., 2007. A serological study of Babesia caballi and Theileria equi in Thoroughbreds in Trinidad. Vet. Parasitol. 144, 167–171. Balkaya, I., Utuk, A.E., Piskin, F.C., 2010. Prevalence of Theileria equi and Babesia caballi in donkeys from Eastern Turkey in winter season. Pak. Vet. J. 30, 245–246. ˝ Barbosa, I.P., Bose, R., Peymann, B., Friedhoff, K.T., 1995. Epidemiological aspects of equine babesioses in a herd of horses in Brazil. Vet. Parasitol. 58, 1–8. Boldbaatar, D., Xuan, X., Battsetseg, B., Igarashi, I., Battur, B., Batsukh, Z., Bayambaa, B., Fujisaki, K., 2005. Epidemiological study of equine piroplasmosis in Mongolia. Vet. Parasitol. 127, 29–32. Brüning, A., 1996. Equine piroplasmosis an update on diagnosis treatment and prevention. Br. Vet. J. 152, 139–151. Brüning, A., Phipps, P., Posnett, E., Canning, E.U., 1997. Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis. Vet. Parasitol. 68, 11–26. Cacciò, S., Cammà, C., Onuma, M., Severini, C., 2000. The beta-tubulin gene of Babesia and Theileria parasites is an informative marker for species discrimination. Int. J. Parasitol. 30, 1181–1185. Camacho, A.T., Guitian, F.J., Pallas, E., Gestal, J.J., Olmeda, A.S., Habela, M.A., Telford III, S.R., Spielman, A., 2005. Theileria (Babesia) equi and Babesia caballi infections in horses in Galicia, Spain. Trop. Anim. Health Prod. 37, 293–302.

255

Chahan, B., Zhang, S., Seo, J., Nakamura, C., Zhang, G., Bannai, H., Jian, Z., Inokuma, H., Tuchiya, K., Sato, Y., Kabeya, H., Maruyama, S., Mikami, T., Xuan, X., 2006. Seroepidemiological evidence for the possible presence of Babesia (Theileria) equi and Babesia caballi infections in donkeys in Western Xinjiang, China. J. Vet. Med. Sci. 68, 753–755. de Waal, D.T., van Heerden, J., 2004. Equine Piroplasmosis. In: Coetzer, J.A.W., Tustin, R.C. (Eds.), Infectious Diseases of Livestock. , second edition. Oxford University Press, New York, pp. 425–434. Donnelly, J., Joyner, L., Graham-Jones, O., Ellis, C., 1980. A comparison of the complement fixation and fluorescent antibody tests in a survey of the prevalence of Babesia caballi in horses in the Sultanate of Oman. Trop. Anim. Health Prod. 12, 50–60. Garcia-Bocanegra, I., Arenas-Montes, A., Hernandez, E., Adaszek, L., Carbonero, A., Almeria, S., Jaen-Tellez, J.A., Gutierrez-Palomino, P., Arenas, A., 2013. Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infection in equids. Vet. J. 195, 172–178. Hailat, N.Q., Lafi, S.Q., Al-Darraji, A.M., Al-Ani, F.K., 1997. Equine babesiosis associated with strenuous exercise: clinical and pathological studies in Jordan. Vet. Parasitol. 69, 1–8. Heuchert, C.M.S., de Giulli Jr., V., de Athaide, D.F., Böse, R., Friedhoff, K.T., 1999. Seroepidemiologic studies on Babesia equi and Babesia caballi infections in Brazil. Vet. Parasitol. 85, 1–11. Ikadai, H., Osorio, C.R., Xuan, X., Igarashi, I., Kanemaru, T., Nagasawa, H., Fujisaki, K., Suzuki, N., Mikami, T., 2000. Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein. Int. J. Parasitol. 30, 633–635. Ikadai, H., Nagai, A., Xuan, X., Igarashi, I., Kamio, T., Tsuji, N., Oyamada, T., Suzuki, N., Fujisaki, K., 2002. Seroepidemiologic studies on Babesia caballi and Babesia equi infections in Japan. J. Vet. Med. Sci. 64, 325–328. Jaffer, O., Abdishakur, F., Hakimuddin, F., Riya, A., Wernery, U., Schuster, R.K., 2010. A comparative study of serological tests and PCR for the diagnosis of equine piroplasmosis. Parasitol. Res. 106, 709–713. Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitology 129 (Suppl.), S3–S14. Kappmeyer, L.S., Perryman, L.E., Hines, S.A., Baszler, T.V., Katz, J.B., Hennager, S.G., Knowles, D.P., 1999. Detection of equine antibodies to Babesia caballi by recombinant B. caballi rhoptry-associated protein 1 in a competitive-inhibition enzyme-linked immunosorbent assay. J. Clin. Microbiol. 37, 2285–2290. Karatepe, B., Karatepe, M., Cakmak, A., Karaer, Z., Ergün, G., 2009. Investigation of seroprevalence of Theileria equi and Babesia caballi in horses in Nigde province, Turkey. Trop. Anim. Health Prod. 41, 109–113. Katz, J.B., Dewald, R., Nicholson, J., 2000. Procedurally similar competitive immuno-assay system for the diagnosis of Babesia equi, Babesia cabali, Trypanosoma equiperdum, and Burkholderia mallei infection in horses. J. Vet. Diagn. Invest. 12, 46–50. Khan, M.Q., Hayat, B., Hayat, C.S., 1987. Prevalence of blood parasites in equines in and around Faisalabad. Pak. Vet. J. 3, 113–116. Knowles, D.P., Perryman, L.E., Kappmeyer, L.S., Hennager, S.G., 1991. Detection of equine antibody to Babesia equi meroziotes protein by a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay. J. Clin. Microbiol. 29, 2956–2958. Knowles Jr., D.P., Kappmeyer, L.S., Stiller, D., Hennager, S.G., Perryman, L.E., 1992. Antibody to a recombinant merozoite protein epitope identifies horses infected with Babesia equi. J. Clin. Microbiol. 30, 3122–3126. Knowles Jr., D.P., 1996. Control of Babesia equi parasitemia. Parasitol. Today 12, 195–198. Kokab, R., 1986. Observations on blood parasites of ungulates of N.W.F.P. (Pakistan). Thesis, Master of Science. Department of Zoology, University of Peshawar, Pakistan. Kouam, M.K., Kantzoura, V., Gajadhar, A.A., Theis, J.H., Papadopoulos, E., Theodoropoulos, G., 2010. Seroprevalence of equine piroplasms and hostrelated factors associated with infection in Greece. Vet. Parasitol. 169, 273–278. Kumar, S., Kumar, A., Sugimoto, C., 2009. A perspective on Theileria equi infections in donkeys. Jpn. J. Vet. Res. 54, 171–180. Kuttler, K.L., 1988. World-wide impact of babesiosis. In: Ristic, M. (Ed.), Babesiosis of Domestic Animals and Man. CRC Press, Boca Raton, Florida, USA, pp. 1–22. Mehlhorn, H., Schein, E., 1998. Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn, Schein 1998. Parasitol. Res. 84, 467–475. Nagore, D., Garcia-Sanmartin, J., Garcia-Perez, A.L., Juste, R.A., Hurtado, A., 2004. Detection and identification of equine Theileria and Babesia species by reverse line blotting: epidemiological survey and phylogenetic analysis. Vet. Parasitol. 123, 41–54. Nuttall, G.H.F., Strickland, C., 1910. The parasites of the horse piroplasmosis response of biliary fever. Zentralb Bakteriol 1, 524–525 [in German].

256

M.H. Hussain et al. / Veterinary Parasitology 202 (2014) 248–256

OIE (World Organization for Animal Health), 2008. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Ch. 2.5.8. Equine Piroplasmosis. http://www.oie.int/fileadmin/Home/eng/Health standards/ tahm/2.05.08 EQUINE PIROPLASMOSIS.pdf (accessed 05.06.13). Radostits, O.M., Gay, C.C., Hinchcliff, K.W., Constable, P.D., 2007. Veterinary Medicine: A textbook of Diseases of Cattle, Horses, Sheep, Pigs and Goat, tenth edition. W. B. Saunders Co., Philadelphia, USA. Rashid, A., Mubarak, A., Hussain, A., 2009. Babesiosis in equines in Pakistan: a clinical report. Vet. Ital. 45, 391–395. Ribeiro, M.F.B., Costa, J.O., Guimaräes, A.M., 1999. Epidemiological aspects of Babesia equi in horses in Minas Gerais, Brazil. Vet. Res. Commun. 23, 385–390. Rüegg, S.R., Torgerson, P., Deplazes, P., Mathis, A., 2007. Age-dependent dynamics of Theileria equi and Babesia caballi infections in southwest Mongolia based on IFAT and/or PCR prevalence data from domestic horses and ticks. Parasitology 134, 939–947. Rüegg, S.R., Heinzmann, D., Barbour, A.D., Torgerson, P.R., 2008. Estimation of the transmission dynamics of Theileria equi and Babesia caballi in horses. Parasitology 135, 555–565. Seifi, H.A., Mohri, M., Sardari, K., 2000. A mixed infection of Babesia equi and Babesia caballi in a racing colt: A report from Iran. J. Eq. Vet. Sci. 20, 858–860. Sevinc, F., Maden, M., Kumas, C., Sevinc, M., Ekici, O.D., 2008. A comparative study on the prevalence of Theileria equi and Babesia caballi infection in horse sub-population in Turkey. Vet. Parasitol. 156, 173–177. Shkap, V., Cohen, I., Leibovitz, B., Savitsky Pipano, E., Avni, G., Shofer, S., Giger, U., Kappmeyer, L., Knowles, D., 1998. Seroprevalence of Babesia equi among horses in Israel using competitive inhibition ELISA and IFA assays. Vet. Parasitol. 76, 251–259.

Sigg, L., Gerber, V., Gottstein, B., Doherr, M.G., Frey, C.F., 2010. Seroprevalence of Babesia caballi and Theileria equi in the Swiss horse population. Parasitol. Int. 59, 313–317. Thompson, P.H., 1969. Ticks as vectors of equine piroplasmosis. J. Am. Vet. Med. Assoc. 155, 454–457. Thrusfield, M., 2005. Veterinary Epidemiology, second edition. Blackwell Science Ltd., U.K. Torina, A., Vicente, J., Alongi, A., Scimeca, S., Tulra, R., Nicosia, S., Di Marco, V., Caracappa, S., de la Fuente, J., 2007. Observed prevalence of tick-borne pathogens in domestic animals in Sicily, Itlay during 2003–2005. Zoonoses Public Health 54, 8–15. Uilenberg, G., 2006. Babesia – a historical overview. Vet. Parasitol. 138, 3–10. Urdaz-Rodriguez, J.H., Fosgate, G.T., Alleman, A.R., Rae, D.O., Donovan, G.A., Melendez, P., 2009. Seroprevalence estimation and management factors associated with high herd seropositivity for Anaplasma marginale in commercial dairy farms of Puerto Rico. Trop. Anim. Health Prod. 41, 1439–1448. Vial, H.J., Gorenflot, A., 2006. Chemotherapy against babesiosis. Vet. Parasitol. 138, 147–160. World Animal Health Information Database (WAHID) Interface, 2013. http://www.oie.int/wahis 2/public/wahid.php/Diseaseinformation/ Diseasetimelines (accessed 05.06.13). Xuan, X., Igarashi, I., Tanaka, T., Fukumoto, S., Nagasawa, H., Fujisaka, K., Mikami, T., 2001. Detection of Babesia equi in horses by a latex agglutination test using recombinant EMA-1. Clin. Diagn. Lab. Immunol. 8, 645–646. Zinora, A., Coombs, D.K., Mohammad, F., Campbell, M.D., Caesar, E., 2007. A serological study of Babesia cabali and Theileria equi in Thoroughbreds in Trinidad. Vet. Parasitol. 144, 167–171.